The main aim of workers in the field of recombinant DNA technology is to achieve as high a level of production as possible of a particular polypeptide. This is particularly true of commercial organisations who wish to exploit the use of recombinant DNA technology to produce polypeptides which naturally are not very abundant.
Generally the application of DNA technology involves the cloning of a gene encoding the desired polypeptide, placing the cloned gene in a suitable expression vector, transfecting a host cell line with the vector, and culturing the transferred cell line to produce the polypeptide. It is almost impossible to predict whether any particular vector or cell line or combination thereof will lead to a useful level of production.
In general, the factors which significantly affect the amount of polypeptide produced by a transferred cell line are: 1. gene copy number, 2. efficiency with which the gene is transcribed and the mRNA translated, 3. the stability of the mRNA and 4. the efficiency of secretion of the protein.
The majority of work directed at increasing expression levels of recombinant polypeptides has focussed on improving transcription initiation mechanisms. As a result the factors affecting efficient translation are much less well understood and defined, and generally it is not possible to predict whether any particular DNA sequences will be of use in obtaining efficient translation.
Attempts at investigating translation have consisted largely of varying the DNA sequence around the consensus translation start signal to determine what effect this has on translation initiation (Kozak M. Cell 41 283-292 (1986)).
Studies involving expression of desired heterologous genes normally use both the coding sequence and at least part of the 5'-untranslated sequence of the heterologous gene such that translation initiation is from the natural sequence of the gene. This approach has been found to be unreliable probably as a result of the hybrid nature of the 5'-untranslated region and the fact that the presence of particular 5-untranslated sequences can lead to poor initiation of translation (Kozak H. Procl. Natl. Acad. Sci. 83 2850-2854 (1986) and Pelletier and Sonenberg Cell 40 515-526 (1985)). This variation in translation has a detrimental effect on the amount of the product produced.
Previous studies (Boshart et al Cell 41 521-530 (1985) and Pasleau et al, Gene 38 227-232 (1985); Stenberg et al, J. Virol 49 (1) 190-199 (1984); Thomsen et al Proc. Natl. Acad. Sci. USA 81 659-663 (1984) and Foecking and Hofstetter Gene 45 101-105 (1986)) have used sequences from the upstream region of the hCMV-MIE gene in expression vectors. These have, however, solely been concerned with the use of the sequences as promoters and/or enhancers. Spaete and Mocarski (J. Virol 56 (1) 135-143, 1985) have used a PstI to PstI fragment of the hCMV-MIE gene encompassing the promoter, enhancer and part of the 5'-untranslated region, as a promoter for expression of heterologous genes. In order to obtain translation the natural 5'-untranslated region of the heterologous gene was used.
In published European Patent Application No. 260148, a method for the continuous production of a heterologous protein is described. The expression vectors constructed contain part of the 5'-untranslated region of the hCMV-MIE gene as a stabilising sequence. The stabilising sequence is placed in the 5'-untranslated region of the gene encoding the desired heterologous protein i.e. the teaching is again that the natural 5'-untranslated region of the gene is essential for translation.